Munc13-4 Regulates Peptide Hormone Degradation in Neuroendocrine Cells

项目摘要 粗面内质网中肽激素的生物合成，高尔基体中分泌颗粒的包装，以及胞吐作用 在质膜上的SG是在神经内分泌细胞中被充分研究的过程。然而， 了解细胞内肽激素降解和SG周转。只有一小部分（<10%） SG储存的肽激素用于生理刺激，使得大多数SG将经历周转。 SG降解机制需要通过SG生物发生来平衡，以维持SG池，目前尚不清楚这是如何实现的。 实现了协调。垂体和内分泌胰腺的经典EM研究表明， 与多泡体（MVB）/溶酶体合并，在称为分泌吞噬的过程中降解。我们开发 在神经内分泌BON细胞中进行几种基于荧光的细胞测定以检测SG-MVB/溶酶体合并。 由于Munc 13 -4参与多种细胞类型的内体融合机制，我们进行了测定 在Munc 13 -4敲低的细胞中观察到强烈的抑制。在aim 1中，我们将扩展结构化 照明显微镜研究，以解决是否crinophagy是主要的机制， 伴随着微自噬。我们将继续进行CgA计时器蛋白的研究，以证明老年SG是 优先降解，并确定这些SG是否被泛素化标记。最后，通过重组 在无细胞测定中SG-MVB/溶酶体融合，我们将确定Munc 13 -4是否直接促进细胞的增殖。 SG-MVB/溶酶体合并。为了证明Munc 13 -4敲低应模拟溶酶体抑制剂， 处理后，我们揭示了Munc 13 -4敲低可能在SG降解的上游具有额外的影响。 我们观察到SG较小，而不是SG蓄积。当仔细检查时，小SG 观察到经历组成性胞吐作用。这表明Munc 13 -4对SG的生物合成也有影响 通路后续研究将包括标记蛋白的免疫荧光研究，以确定哪里小 在Munc 13 -4敲低的细胞中出现SGs。Munc 13 -4敲低的总体表型由一个 从SG降解转变为组成性胞吐。如果这对激素或激素原是真的， 将具有我们将在Munc 13 -4 KO小鼠中评估的生理后果。在目标2中，我们将确定 Munc 13 -4调节SG-MVB/溶酶体融合的机制基础。使用siRNA筛选， SG-MVB/溶酶体融合的稳健的基于荧光的测定，我们将鉴定Rab、SNARE和辅助 SG-MVB融合所需的因子。我们将开发一种基于脂质体的融合检测方法， 在屏幕上。这将使我们能够测试哪些因子如Rab 27调节Munc 13 -4依赖性融合。最后，随着 高度纯化的Munc 13 -4蛋白，我们将通过冷冻电镜确定其结构。基于脂质体融合 通过测定，我们将确定Munc 13 -4在冷冻ET研究中的束缚性质。总的来说，这项工作将揭示 Munc 13 -4在神经内分泌细胞中重要的新细胞作用，并有助于对 Munc 13 -4的束缚角色

PROJECT SUMMARY Peptide hormone biosynthesis in the RER, packaging into secretory granules (SGs) in the Golgi, and exocytosis of SGs at the plasma membrane are well-studied processes in neuroendocrine cells. However, there is limited understanding of intracellular peptide hormone degradation and SG turnover. Only a small fraction (<10%) of SG-stored peptide hormones are utilized in physiological stimulation so that most SGs would undergo turnover. SG degradation mechanisms need to be balanced by SG biogenesis to maintain SG pools it is unclear how this coordination is achieved. Classical EM studies in the pituitary and endocrine pancreas had shown that SGs merge with multivesicular body (MVB)/lysosomes for degradation in a process termed crinophagy. We developed several fluorescence-based cell assays in neuroendocrine BON cells to detect the SG-MVB/lysosome merge. Because Munc13-4 is involved in endosomal fusion mechanisms in multiple cell types, we conducted the assays in cells knocked down for Munc13-4 and observed strong inhibition. In aim 1, we will extend structured illumination microsope studies to resolve whether crinophagy is the major mechanism and whether it is accompanied by microautophagy. We will pursue CgA timer protein studies to document that older SGs are preferentially degraded and determine whether such SGs are marked by ubiquitination. Lastly, by reconstituting SG-MVB/lysosome merge in a cell-free assay, we will determine whether Munc13-4 acts directly to promote the SG-MVB/lysosome merge. In efforts to document that Munc13-4 knockdown should mimic lysosome inhibitor treatment, we revealed that Munc13-4 knockdown has additional effects likely upstream from SG degradation. Instead of SG accumulation, we observed that SGs were smaller. When examined carefully, the small SGs were observed to undergo constitutive exocytosis. This implies that Munc13-4 also has effects on the SG biogenesis pathway. Follow-up studies will include immunofluorescense studies of marker proteins to determine where small SGs emerged in Munc13-4 knockdown cells. The overall phenotype of Munc13-4 knockdown is dominated by a shift from SG degradation to constitutive exocytosis. Were this to be true for hormones or prohormones this would have physiological consequences that we will assess in a Munc13-4 KO mouse. In aim 2, we will determine the mechanistic basis for the Munc13-4 regulation of the SG-MVB/lysosome merge. An siRNA screen using a robust fluorescence-based assay for the SG-MVB/lysosome merge, we will identify Rab, SNARE, and accessory factors required for SG-MVB fusion. We will develop a liposome-based fusion assay using the proteins identified in the screen. This will allow us to test what factors like Rab27 regulate Munc13-4-dependent fusion. Lastly, with highly purified Munc13-4 protein we will determine its structure by cryo-EM. Based on a on a liposome fusion assay, we will determine the tethering properties of Munc13-4 in cryo-ET studies. Overall this work will reveal important new cellular roles for Munc13-4 in neuroendocrine cells and contribute a molecular understanding on the tethering role for Munc13-4.